Certain proteins, when introduced into an organism, have been shown to specifically localize to and bind to target sites. These target-specific proteins can be used as carriers of diagnostic and therapeutic agents, allowing such agents a means of specifically localizing to the target. The use of target-specific proteins as carriers involves the binding of the diagnostic or therapeutic agent to the protein; forming a conjugate which combines the diagnostic or therapeutic properties as well as the localization properties of the protein.
The use of monoclonal antibodies and antibody fragments as target-specific carriers of diagnostic or therapeutic agents should ideally provide an efficient means of localizing such agents to target tissue. Monoclonal antibodies are highly specific and can be used, for example, for imaging specific target sites or as vehicles to deliver substances to target sites. In recent years numerous monoclonal antibodies have been developed with affinity for targets such as atherosclerotic tissue, fibrinogen, myosin and tumors, to name just a few, and work in this area continues. The attachment of radiometals to proteins, especially antibodies and antibody fragments, results in the formation of new radiodiagnostic and radiotherapeutic agents. The performance of the radiometal-protein conjugates depends on a number of factors, such as the stability of the radiometal-protein bond and the ability of the conjugates to localize to the target tissue.
Proteins and antibodies have been shown to form stable bonds to radiometals by the use of bifunctional coupling agents. The bifunctional agent is selected such that it is capable of binding radiometals by chelation and also form a stable linkage to the protein. Thus, the protein or antibody is bound to the radiometal through the bifunctional coupling agent. For example, diethylenetriaminepentaacetic acid (DTPA) has been conjugated onto an antimyosin antibody, and the protein-bound DTPA used to chelate indium-111 (Khaw, et al., Science, 209, 295-97 (1980). See also Krejcare, et al., Biochem. Biophys. Res. Comm., 77, 581-85 (1977) and Childs, R. L. and Hnatowich, D. J., J. Nucl. Med., 26, 293 (1985)). This approach has also been used where particular diaminodithiol and diamidedithiol chelating agents have been coupled to antibodies (Fritzberg, et al., J. Nucl. Med., 27, 957-58 (1986), Eary, J. et al., J. Nucl. Med., 28, 650-51 (1987) and A. R. Fritzberg, et al., Proc. Nat. Acad. Sci. USA, 85, 4025-29 (1988)). Chelated radiometals and bifunctional coupling agents have been linked to proteins by lysyl side chain amino groups (EPO Publication No. 188, 256). Chelators have also been site selectively attached to oxidized antibody carbohydrate moieties (EPO Publication No. 173, 629, U.S. Pat. No. 4,671,958). Chelators can also be attached by reaction with free sulfhydryl groups (U.S. Pat. No. 4,659,839, U.S. Pat. No. 4,671,958 and EPO Publication No. 173, 629).
Useful thiol-containing bifunctional agents for the attachment of radionuclides to proteins have been disclosed (pending U.S. Ser. No. 199,931, filed Jun. 15, 1988 and pending U.S. Ser. No. 235,999, filed Aug. 24, 1988). These disclosures demonstrate the usefulness of sulfhydryl-selective attachment of bifunctional agents to protein sulfhydryls and the subsequent removal of thiol protecting groups from the chelating portion of the bifunctional agents. Also demonstrated was a reduction of radionuclide accumulation in the excretory organs by the use of cleavable linking moieties. These cleavable linking moieties are used to join the chelating portion and the sulfhydryl-selective portion of the bifunctional agents.
Antibodies and antibody fragments have also been used as target specific carriers of toxins and drugs for therapeutic purposes. For example, the polypeptide toxin Ricin consists of two peptide chains. The A chain is toxic to cells, and can be covalently linked to antibodies or antibody fragments and specifically delivered to target cells (I. Pastan, M. C. Willingham and D. J. P. Fitzgerald, Cell, 47, 641-48 (1986)). Antibodies have also been used as specific targeting carriers of a variety of antineoplastic agents (D. C. Edwards, Pharmac. Ther., 23, 147-77 (1983)).
The utility of a target-specific protein conjugate can be enhanced by the addition of increasing amounts of an agent to the protein. For example, the addition of multiple chelating sites allows greater specific activities per protein molecule by binding larger numbers of radionuclides. Antibodies and antibody fragments have been shown to be sensitive to the number of chelators directly bound to them (Childs, R. L. and Hnatowich, D. J., J. Nucl. Ned., 26, 293 (1985)). Multiple chelating sites have been attached to a single site on an antibody by the use of a carrier (B. A. Khaw, et al., J. Nucl. Med., 27, 909-10 (1986); Y. Manabe, C. Longley and P. Furmanski, Biochim. Biophys. Acta, 883, 460-67 (1986); V. P. Torchilin, et al., Hybridoma. 6, 229-40 (1987)). This approach involves the attachment of many diethylenetriaminepentaacetic acid moieties to poly-lysine, resulting in a net negative charge of the poly-lysine carrier. The antibodies and antibody fragments so modified were observed to localize in the liver, with an increase in blood clearance rates.
Clearance of small proteins from the blood in vivo occurs largely in the kidneys. Movement across the glomerular capillary barrier is dependant on both size and the charge of the protein (B. M. Brenner, T. H. Hostetter and H. D. Humes, Am. J. Physiol., 234, F455-60 (1987)). Increasing the positive charge (increasing pI) is known to facilitate glomerular filtration. However, a study of cationically modified autologous albumin found that a decreased accumulation in the urine occurred (T. Z. Coimbra, M. R. Furtado, J. J. Lachat and I. F. de Carvalho, Nephron, 33, 208-15 (1983)).
Means have been found for preparing stable protein-radiometal conjugates which localize to target tissue. The distribution characteristics in vivo of the protein-radiometal conjugates have not been modified in a controllable fashion. For example, the in vivo tumor to blood ratio of a specific monoclonal antibody was lower for iodination (I-131), but independent of labeling using Tc-99m, Se-75 and In-111 (A. Steinstrasser, et al., J. Nucl. Med., 28, 693 (1987)). D. A. Goodwin states (J. Nucl. Med., 28, 1358-62 (1987)) that although antibody fragments (Fab and F(ab').sub.2) are able to diffuse more rapidly into tumors than whole antibodies, absolute tumor concentrations will be lower for the fragments relative to the whole antibody due to lower integral blood concentrations. From this, the conclusion can be drawn that a more effective radioimmunotherapeutic agent would need the diffusion characteristics of an antibody fragment, yet would also need an increased blood residence time to achieve greater tumor accumulation.